ABSTRACT
Background Human Tenon fibroblasts (HTFs) are the main cell component in scar tissue after antiglaucomatous filtrating surgery.To study the in vitro growth and proliferation features will offer an approach to the preventing and treating of scarring following antiglaucomatous filtrating surgery.Objective The goal of present study was to investigate the growth characteristics of HTFs in vitro from patients with bleb scarring after antiglaucomatous filtrating surgery.Methods This study process was approved by Ethic Committee of Beijing Chaoyang Hospital,and informed consent was obtained from each subject prior to the initaion of the study.The specimens of scaring tissue were obtained during the secondary trabeculectomy from 8 eyes with bleb scaring after initial antiglaucomatous filtrating surgery with the tissue size of 4 mm×5 mm,and the normal Tenon capsule specimens were acquired during the strabismus surgery from 8 eyes in the same way.HTFs were primarily cultured and passaged by tissue explants adherent method and identified using immunoinfluorescence technique with vemintin antibody.Nest PCR was used to exclude the mycoplasma infection during the culturing process.The morphology,growth curve and population doubling time (PDT) of the fifth generation of cells were examined and calculated by luminescence method cell vitality method 0,4,7,11,14 days and compared between the patients and normal groups.The stability of the cell growth was assessed by comparing the morphology,growth curve and PDT between the fifth generation and fifteenth generation of HTFs.Results Primarily cultured cells reached confluence 1-2 weeks with the similar shape to HTFs.The growth properties of the cells were same between the scarring group and normal group and showed positive response for vemintin antibody.No react band for mycoplasma was detected.The PDT was (20.54±3.51) hours and (18.86±2.91) hours in the scarring group and normal group,respectively,showing insignificant difference between them (t=0.634,P=0.561).No significant differences were found in the number of cells in 4,7,11 and 14 days after passage (t =2.663,P =0.081; t =0.194,P =0.863 ; t =3.338,P =0.053 ; t =0.627,P =0.565),with a consistent growth curve with the lapse of time between the two groups.The HTFs from scarred Tenon capsule fibrosis were cultured and passaged until the fifteenth generation.The PDT was gained to be (20.54 ±3.51) hours in the fifth generation of cells and (22.84±4.15) hours in the fifteenth generation of cells,without significant difference between them (t =0.733,P =0.505).The number of cellsin 4,7,11 and 14 days was not significantly different between the fifteenth generation and the fifth generation of cells (t=1.528,P=0.235;t=0.269,P=0.786;t=1.954,P=0.139;t =0.730,P =0.506).In addition,a good and stable growth curve also was exhibited in the fifteenth generation of cells compared with the fifth generation of cells.Conclusions Bleb scar-derived HTFs present good and stable growth in vitro.This result demonstrates HTFs were target cells in antifibrosis study after antiglaucoma filtrating surgery.
ABSTRACT
Background The excessive growth of human Tenon fibroblasts (HTFs) is a primary cause of failure of anti-glaucomatous filtering surgery.To seek a drug of anti-fibrosis is of an important significance for improving the successful rate of anti-glaucomatous filtration surgery.Objective The goal of this study was to investigate the effect of paclitaxel on proliferation of HTFs in vitro.Methods Human Tenon tissue was obtained during the anti-glaucomatous filtering surgery.HTFs were cultivated using explant method and 3-6 generations of cells were used in the experiment.The cells were identified by immunochemistry using keratin,vimentin,fibronectin and S-100.Different concentrations (0,1 ×10-s,1 × 10-7,1 × 10-6 mol/L) of paclitaxel were added into the medium,and then the cell indexes (CI) in the various groups were detected by real-time cell electronic sensing (RT-CES) 24 hours after affection of paclitaxel.Apoptosis of the cells was examined using DAPI staining,and the proportion of the cells in different cycles were assayed by flow cytorneter 12 hours after addition of paclitaxel.The expressions of matrix metalloproteinase-1 (MMP-1) protein and mRNA were detected by ELISA and real-time PCR,respectively.Results The cells migrated from the cultivated tissue within 7 days with the fibrocyte-like shape.The cells showed the positive response for vimentin and absent response for keratin,fibronectin and S-100.The CI values were 1.093 ±0.191,0.665 ± 0.093 and 0.473 ± 0.117 in the 1 × 10-8,1 × 10-7 and 1 × 10-6 mol/L paclitaxel groups,showing significant rise in comparison with the 1.514 ±0.283 of the 0 mol/L paclitaxel group (all at P =0.000).The cell nuclei were normal in the 0 mol/L paclitaxel group,however,blue-fluorescent particles and apoptotic bodied were found in the cell nuclei after affection of paclitaxel.The proportion of G2/M phase of cells were (9.20±0.80) ％,(12.37±0.45)％ and (13.80±0.35)％ in the 1×10-8 mol/L,1×10-7 mol/L and 1×10-6 mol/L paclitaxel groups,which were higher than the (7.17±0.50) ％ in the 0 mol/L paclitaxel group (P=0.005,0.000,0.000).In addition,the relative expressing level of M MP-1 mRNA (MMP-1 mRNA/GAPDH mRNA) and the expression level of MMP-1 protein in the HTFs were significantly lower in the 1 ×10-8 mol/L,1 × 10-7 mol/L and 1 × 10-6 mol/L paclitaxel groups than those in the 0 mol/L group (all at P＜0.05).Conclusions Paclitaxel at the concentrations of 1 × 10-8 mol/L-1 × 10-6 mol/L inhibits the proliferation of HTFs in vitro by arresting the cellular mitosis and inducing cell apoptosis.These effects probably associated with down-regulation of MMP-1 expression in the HTFs.